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PubMed1988

Approach to maceration mechanism in enzymatic pulping of bast fibers by alkalophilic pectinolytic enzymes produced by Erwinia species.

Tissue maceration was generally elucidated by the action of endo-polygalacturonase and endo-pectate or -pectin lyase (endo-PAL or -PNL). In a process of screening of Erwinia and Pseudomonas strains for enzymatic pulping of pectocellulosic bast fibers, it was found that their PAL productivity was not completely related with defibration activity, i.e., the fact that an E.chrysanthemi strain showed high PAL productivity but possessed rather low defibration activity. Moreover, defibration activity was parallel to the amount of neutral sugars released during pulping. Based on these fact, the maceration or enzymatic pulping of basts was estimated to proceed not only by cleavage of interfiber bonding cause by PAL action but also another factors. Among three possibilities proposed on the maceration mechanism of basts, it was elucidated by a concerted action of PAL and PNL with an aid of xylanase. In addition, a quantitative determinative method of maceration activity toward basts was also presented.

Journal Article↗
PubMed2005

Structure-immunomodulating activity relationships of a pectic arabinogalactan from Vernonia kotschyana Sch. Bip. ex Walp.

Structure and immunological characteristics of the pectic arabinogalactan Vk2a (previously reported as Vk100A2a) from the roots of Vernonia kotschyana Sch. Bip. ex Walp. were investigated after enzymatic digestion of the galacturonan moiety and the side chains of the rhamnogalacturonan structure of Vk2a. endo-alpha-D-(1-->4)-Polygalacturonase digestion released the high molecular weight 'hairy region' (Vk2a-HR) and oligogalacturonides. Vk2a-HR consisted of GalA (4-linked) and Rha (2- or 2,4-linked) in a 1:1 ratio, with 60% of Rha branched at C-4. The Rha located in the rhamnogalacturonan core was branched randomly by Gal units. Vk2a-HR was rich in neutral sugars such as Araf 5- (12.2%) and 3,5-substituted (12.8%) and terminally- (14.1%) linked and Gal 4- (13.0%), 3- (0.9%), 6- (2.2%) and 3,6- (1.1%) substituted. Arabinans with chain lengths up to 11 units were identified. Araf residues were attached to C-3 of alpha-L-(1-->5)-Araf chains and to C-4 of Gal residues. Single Gal units and chains of beta-D-(1-->6)-linked galacto di- to penta-saccharides were attached to a beta-D-(1-->3)-galactan core. All the enzyme resistant fractions expressed potent complement fixation and induction of B-cell mitogenic activity, and the present study indicates that there may be several and possibly structurally different active sites involved in the bioactivity of Vk2a. The bioactive sites may be located both in the more peripheral parts of the molecule but also in the inner core of the 'hairy region' or in larger enzyme-resistant chains.

Animals↗
PubMed2006

Influence of ultraviolet-C on the compositions of cell-wall polysaccharides and carbohydrase activities of Silene vulgaris callus.

UV-C irradiation (254 nm) was found to enhance the secretion of some cell-wall-degrading enzymes, especially the following carbohydrases: beta-galactosidase, alpha-L-arabinofuranosidase, polygalacturonase, pectinesterase, cellulase, xylanase, and beta-xylosidase, in the campion callus, contributing thereby to an alteration in the polysaccharide structure. The relative amounts of the galactose and arabinose residues in pectin (silenan) and of arabinose in arabinogalactan of calli irradiated during the exponential phase were shown to decrease during the stationary phase. A decrease in the degree of SV methylesterification was found for the irradiated callus. These alterations were found to persist over a long period of culturing time. Decreasing the relative amounts of the arabinose residues in arabinogalactan and pectin and the galactose residues in silenan corresponded to increasing activity of alpha-L-arabinofuranosidase and beta-galactosidase, respectively, due to treatment with UV-C. UV-C irradiation may be used as a tool for modifying the structural features of the cell-wall polysaccharides, such as the relative amounts of galactose and arabinose residues in the side chains of polysaccharides, with the purpose of obtaining physiologically active polysaccharides with the desired properties and structural features.

Cell Wall↗
PubMed2005

Separation of pectin methylesterase isoenzymes from tomato fruits using short monolithic columns.

One of the main forms of tomato pectin methylesterase (PME; EC 3.1.1.1.1) that is applicable to the food industry was isolated from fresh tomato fruit. The extraction of the PME isoenzymes involved washing the fresh tomato flesh with water in order to remove sugars and than solubilizing the enzymes with a diluted HCl solution at pH 1.6. The extract was then neutralized to pH 7.4 using buffer solution. After filtration, the solution was directly fractioned using Convective Interaction Media (CIM) short monolithic disk column bearing sulfonyl (SO3) groups and using a linear gradient from 0 to 700 mM NaCl. The injection volume was 3 ml and the diameter of the column was 12 mm and length 3 mm. The isolated fractions were monitored for protein content and PME activity. The fraction with the targeted enzyme, which showed NaCl independent activity, was further purified and concentrated by ultrafiltration and finally purified by a second semi-preparative cation-exchange chromatography step using a CIM carboxymethyl (CM) disk monolithic column consisting of two disks and applying a step gradient. From 1 kg of fresh tomato fruits, 7.5 mg of purified PME with molecular mass estimated to be 26 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was obtained. A fraction with mixed PME and polygalacturonase activity was also obtained. Compared to the published procedures for the isolation and purification of PME from plant materials, this new procedure is much faster and more efficient. The potential application of CIM disk short monolithic columns in the analysis and semi-preparative extraction and isolation of the PME isoenzyme is presented.

Carboxylic Ester Hydrolases↗
PubMed2006

Safety evaluation of pigment containing Aspergillus carbonarius biomass in albino rats.

Aspergillus carbonarius, an ascomycetes fungus, is known to produce pectinase in solid-state fermentation. A mutant strain of A. carbonarius UV-10046 selected for temperature tolerance over produced polygalacturonase and during growth accumulated an yellow pigment in its biomass. Since the colored fungus suggested its application for food use, the freeze-dried biomass was evaluated to assess its safety in experimental animals. Acute and sub-acute toxicity studies were conducted on both sexes of albino rats. Feeding acute doses of A. carbonarius freeze-dried biomass at 0.5-5.0g/kg body weight to adult rats did not show any symptoms of toxicity or mortality of the rats. Similarly, dietary feeding of A. carbonarius at 0.25-2.0% level (w/w) for 14 weeks did not produce any significant changes in food intake or gain in body weight of the experimental rats compared to control rats. There were no significant differences in the relative weight of vital organs, hematological parameters, macroscopic and microscopic changes in vital organs and serum enzyme levels between the experimental and control groups. The results clearly showed that acute and sub-acute oral feeding of freeze-dried whole cells of A. carbonarius mutant for 14 weeks did not produce any toxic effects in male and female rats.

Administration, Oral↗
PubMed2005

Characterization of Lactobacillus strains and monitoring by RAPD-PCR in controlled fermentations of "Almagro" eggplants.

The characterization of 23 Lactobacillus strains was performed. The strains were assayed for biogenic amine-forming capacity, hydrogen peroxide production, pectin esterase, cellulase and polygalacturonase production, growth rate, acidifying capacity and salt tolerance. Three strains were selected which belonged to the species, Lactobacillus brevis, Lactobacillus plantarum and Lactobacillus fermentum. Different starter cultures prepared as combinations of these three strains were assayed in pilot scale fermentations and Randomly Amplified Polymorphic DNA (RAPD) analysis, using a previously selected random primer, was applied for monitoring the inoculated strains. The course of fermentations was similar in all batches but sensorial analysis of eggplants fermented using a mixed culture of the three strains displayed the best results, and no differences were obtained when compared with commercial eggplants.

Biogenic Amines↗
PubMed2005

A possible signal transduction pathway for cyclin D2 expression by a pectic polysaccharide from the roots of bupleurum falcatum L. in murine B cell.

Bupleuran 2IIc, a pectic polysaccharide isolated from the roots of bupleurum falcatum L., was previously characterized as a T-cell-independent B cell mitogen. This study focuses on elucidating the mechanism by which bupleuran 2IIc induces cyclin D2 production for inducing mitogenesis in murine B cells. Bupleuran 2IIc was digested with endo-alpha-(1-->4)-D-polygalacturonase and the resulting bupleuran 2IIc/PG-1 ("ramified" region) strongly stimulated cyclin D2 expression. When murine B cells were stimulated with bupleuran 2IIc/PG-1, phosphorylation of tyrosine residues of a number of proteins was observed. Cyclin D2 expression by bupleuran 2IIc/PG-1 was inhibited by the tyrosine kinase inhibitors, genistein and herbimycin A, and the Src family tyrosine kinase inhibitor, PP2, suggesting a possible role for tyrosine kinases. The stimulation by bupleuran 2IIc/PG-1 of cyclin D2 expression was significantly decreased by inhibitors, PI 3-kinase (LY294002 and Wortmannin), PLCgamma (U73122), PKC (H-7), receptor-operated calcium entry inhibitor (SK&F 96365), and calcineurin (FK506). Both PD98059 and U0126, highly selective inhibitors of MEK1 and MEK1/2, respectively, did not strongly suppress the expression of cyclin D2 after stimulation by bupleuran 2IIc/PG-1. The results suggest that (1) bupleuran 2IIc/PG-1 is the active site for induction of cyclin D2 by bupleuran 2IIc, (2) the expression of the cyclin D2 gene by bupleuran 2IIc/PG-1 may be mediated via the activation of PI 3-kinase and PLCgamma followed by activation of PKC and calcium mobilization, and (3) the ERK1/2 cascade is not a central signaling pathway for bupleuran 2IIc/PG-1-induced cyclin D2 expression.

Animals↗
PubMed2005

Mapping sugar beet pectin acetylation pattern.

Homogalacturonan-derived partly methylated and/or acetylated oligogalacturonates were recovered after enzymatic hydrolysis (endo-polygalacturonase+pectin methyl esterase+side-chain degrading enzymes) of sugar beet pectin followed by anion-exchange and size exclusion chromatography. Around 90% of the GalA and 75% of the acetyl groups present in the initial sugar beet pectin were recovered as homogalacturonan-derived oligogalacturonates, the remaining GalA and acetyl belonging to rhamnogalacturonic regions. Around 50% of the acetyl groups present in sugar beet homogalacturonans were recovered as partly methylated and/or acetylated oligogalacturonates of degree of polymerisation 5 whose structures were determined by electrospray ionization ion trap mass spectrometry (ESI-IT-MSn). 2-O-acetyl- and 3-O-acetyl-GalA were detected in roughly similar amounts but 2,3-di-O-acetylation was absent. Methyl-esterified GalA residues occurred mainly upstream 2-O-acetyl GalA. Oligogalacturonates containing GalA residues that are at once methyl- and acetyl-esterified were recovered in very limited amounts. A tentative mapping of the distribution of acetyl and methyl esters within sugar beet homogalacturonans is proposed. Unsubstituted GalA residues are likely to be present in limited amounts (approximately 10% of total GalA residues), due to the fact that methyl and acetyl groups are assumed to be most often not carried by the same residues.

Acetylation↗
PubMed2000

Analysis of pectic epitopes recognised by hybridoma and phage display monoclonal antibodies using defined oligosaccharides, polysaccharides, and enzymatic degradation.

The structure of epitopes recognised by anti-pectin monoclonal antibodies (mAbs) has been investigated using a series of model lime-pectin samples with defined degrees and patterns of methyl esterification, a range of defined oligogalacturonides and enzymatic degradation of pectic polysaccharides. In immuno-dot-assays, the anti-homogalacturonan (HG) mAbs JIM5 and JIM7 both bound to samples with a wide range of degrees of methyl esterification in preference to fully de-esterified samples. In contrast, the anti-HG phage display mAb PAM1 bound most effectively to fully de-esterified pectin. In competitive inhibition ELISAs using fully methyl-esterified or fully de-esterified oligogalacturonides with 3-9 galacturonic acid residues, JIM5 bound weakly to a fully de-esterified nonagalacturonide but JIM7 did not bind to any of the oligogalacturonides tested. Therefore, optimal JIM5 and JIM7 binding occurs where specific but undefined methyl-esterification patterns are present on HG domains, although fully de-esterified HG samples contain sub-optimal JIM5 epitopes. The persistence of mAb binding to epitopes in pectic antigens, with 41% blockwise esterification (P41) and 43% random esterification (F43) subject to fragmentation by endo-polygalacturonase II (PG II) and endo-pectin lyase (PL), was also studied. Time course analysis of PG II digestion of P41 revealed that JIM5 epitopes were rapidly degraded, but a low level of PAM1 and JIM7 epitopes existed even after extensive digestion, indicating that some HG domains were more resistant to cleavage by PG II. The chromatographic separation of fragments produced by the complete digestion of P41 by pectin lyase indicated that a very restricted population of fragments contained the PAM1 epitope while a (1-->4)-beta-D-galactan epitope occurring on the side chains of pectic polysaccharides was recovered in a broad range of fractions.

Antibodies, Monoclonal↗
PubMed2002

Fluorescent labeling of pectic oligosaccharides with 2-aminobenzamide and enzyme assay for pectin.

Oligogalacturonides [oligomers composed of (1-->4)-linked alpha-D-galactosyluronic acid residues] with degrees of polymerization (DP) from 1 to 10, and a tri-, penta-, and heptasaccharide generated from the backbone of rhamnogalacturonan I (RG-I) were labeled at their reducing ends using aqueous 2-aminobenzamide (2AB) in the presence of sodium cyanoborohydride in over 90% yield. These derivatives were analyzed by high-performance anion-exchange chromatography (HPAEC) and structurally characterized by electrospray-ionization mass spectrometry (ESIMS) and by 1H and 13C NMR spectroscopy. The 2AB-labeled oligogalacturonides and RG-I oligomers are fragmented by endo- and exo-polygalacturonase and by Driselase, respectively. 2AB-labeled oligogalacturonide is an exogenous acceptor for galacturonosyltransferase of transferring galacturonic acid from UDP-GalA. Thus, the 2AB-labeled oligogalacturonides and RG-I oligomers are useful for studying enzymes involved in pectin degradation and biosynthesis and may be of value in determining the biological functions of pectic fragments in plants.

Carbohydrate Sequence↗
PubMed1997

Rhamnogalacturonan I from xylem differentiating zones of Cryptomeria japonica.

Rhamnogalacturonan I (RG-I) was isolated from the pectin of the cell wall of xylem differentiating zones of sugi (Cryptomeria japonica D. Don, gymnosperm) by digesting with endo-alpha-1,4-polygalacturonase followed by gel-permeation chromatography. Structure of RG-I was characterized by glycosyl composition and glycosyl linkage analyses, partial acid hydrolysis, and lithium (Li)-treatment in ethylenediamine. The oligosaccharides produced by partial acid hydrolysis showed the backbone of RG-I to be a disaccharide repeating unit of --> 4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->. Arabian and oligogalactosyl side chains were attached at O-4 of rhamnosyl residues of the backbone. Glycosyl composition and glycosyl linkage analyses suggested that the structures and/or the distribution of the arabinan sidechains of sugi RG-I were different from those of sycamore (angiosperm) RG-I.

Carbohydrate Conformation↗
PubMed2004

The effect of endogenous mRNA levels on co-suppression in tomato.

Introduction of truncated polygalacturonase (PG) transgenes into tomato plants caused the production of small interfering RNAs (siRNAs) and co-suppression of both the endogenous and PG transgenes in ripening fruits by post-transcriptional gene silencing. In order to test the possible effect on co-suppression of the endogenous PG mRNA level, we transferred the PG transgenes from a PG-silenced line (wild type background) by crossing to two ripening regulatory mutants with reduced PG: Never-ripe (Nr, approximately 10% endogenous PG mRNA compared to wild type) and ripening-inhibitor (rin, approximately 1% endogenous PG mRNA) and to wild type (as a control). The PG transgenes caused strong co-suppression of the transgenes and the endogenous PG gene in cells with high PG mRNA background (wild type) and silencing appeared to be linked with higher transgene copy number and/or a particular transgene locus. In cells with low endogenous PG mRNA accumulation (Nr), the endogenous PG gene was preferentially suppressed compared to the transgenes, whose expression was not reduced significantly. The expression of the transgenes was also not reduced in the very low PG background (rin), in which endogenous PG was barely detectable. In all the analysed lines with all three PG background levels, siRNAs accumulated in leaves and green fruits, in which the endogenous PG gene is not transcribed. The relatively abundant production of siRNAs in most of the lines was linked with a particular transgene insert. These results suggest that a certain threshold level of endogenous PG mRNA is required for the co-suppression of the truncated PG transgenes and the endogenous PG gene or for extensive silencing of the transgenes.

Crosses, Genetic↗
PubMed1997

Diet and carbohydrate digestion in the yellow-spotted longicorn beetle Psacothea hilaris.

Larval and adult Psacothea hilaris feed on mulberry wood and leaves, respectively. High levels of endogenous activity against the major dietary carbohydrates, cellulose, hemicellulose, starch and soluble sugars were secreted in the gut of larvae and adults. Activity against pectin was also high and multiple polygalacturonase (EC 3.2.1.15) components were secreted in the gut of larvae. One glycanase component, beta-EG1, which was primarily an endo-beta-1,4-glucanase (EC 3.2.1.4) and another, beta-EG2, which was mostly an endo-beta-1,4-xylanase (EC 3.2.1.8), were also secreted, while at least four additional components hydrolysed laminarin, lichenin and crystalline cellulose. The beta-glycosidase component beta-GD1 was associated with most of the beta-mannosidase (EC 3.2.1.25) and beta-xylosidase (EC 3.2.1.37) activity secreted in the gut of larvae, while another, beta-GD2, was a beta-glucosidase (EC 3.2.1.21), the activity of which was directed against cellobiose and other beta-linked disaccharides, and a beta-fucosidase (EC 3.2.1.38). A beta-galactosidase (EC 3.2.1.23), which did not hydrolyse lactose, was also secreted, as were distinct beta-N-acetylhexosaminidase (EC 3.2.1.52), trehalase (EC 3.2.1.28), alpha-L-arabinosidase (EC 3.2.1.55), alpha-galactosidase (EC 3.2.1.22) and a minimum of four alpha-glucosidase (EC 3.2.1.20) components, one of which was also likely to be associated with a peak of alpha-mannosidase (EC 3.2.1.24) activity. The alpha-glucosidase components varied in their specificity for alpha-linked disaccharides, but none was active against sucrose, which was hydrolysed by a beta-fructofuranosidase (EC 3.2.1.26) component. Overall average levels of activity in larvae were twice those of adults, but the secretion of individual carbohydrases in both was not regulated in response to the relative abundance of particular carbohydrate components in their respective diets.

Journal Article↗
PubMed1998

Rhamnogalacturonan II from cell walls of Cryptomeria japonica.

The pectic polysaccharides isolated from cell walls of xylem-differentiating zones of sugi (Cryptomeria japononica) were degraded with endo-alpha-(1-->4)-polygalacturonase and the polysaccharides, composed mainly of rhamnogalacturonan II (RG-II), were obtained from the degradation products. These polysaccharides consisted of rhamnosyl, fucosyl, arabinosyl, xylosyl, galactosyl, glucosyl, galacturonic acid, glucuronic acid and the characteristic sugars of RG-II, namely, 2-O-methylfucose, 2-O-methylxylose, apiose, aceric acid and thiobarbituric acid assay-positive glycosyl ¿probably, 3-deoxy-D-manno-2-octulosonic acid (Kdo) and 3-deoxy-D-lyxo-heptulosaric acid (Dha)¿. The polysaccharides contained the glycosyl residues of RG-II, besides small amounts of the glycosyl linkages of RG-I. The RG-II was structurally analysed by partial acid hydrolysis and lithium treatment in ethylenediamine. The glycosyl sequences of three compounds generated by partial hydrolysis were not identical to the partial structure of the sycamore (Acer pseudoplatanus) RG-I and RG-II structures previously proposed by Albersheim et al. Furthermore, five novel glycosyl sequences were detected in the products from lithium treatment. The results suggest that the structure of sugi RG-II is somewhat different from that of sycamore RG-II.

Carbohydrate Sequence↗
PubMed1997

The xylose-rich pectins from pea hulls.

The hot acid extract of pea hull, HSP, was rich in galacturonic acid, arabinose and xylose. It was fractionated by copper precipitation followed by ion-exchange chromatography. The copper-soluble fraction represented 26% of HSP and was mostly composed of an arabinan with a low degree of branching, some heteroxylans and a glucan, probably starch. The copper-precipitate (74% of HSP) contained pectins and some residual arabinan, xylan, glucan and mannan. One of the pectic fractions was rich in terminal xylose and fucose; it could be partially degraded by endo-polygalacturonase but not by endo-xylanase and seemed to contain xylogalacturonans.

Carbohydrate Conformation↗
PubMed2000

Effects of a pectic polysaccharide from a medicinal herb, the roots of Bupleurum falcatum L. on interleukin 6 production of murine B cells and B cell lines.

The effect of T-cell-independent B cell mitogen, a pectic polysaccharide, bupleuran 2IIc, from a medicinal herb, the roots of Bupleurum falcatum L. on interleukin 6 (IL-6) production of murine B cells and B cell lines was investigated in order to clarify the mechanism of enhanced immunoglobulin (Ig) secretion from B cells. Bupleuran 2IIc enhanced IgM secretion from highly purified murine normal B cells. When normal B cells from murine spleen were cultured with bupleuran 2IIc in the presence of anti-IL-6 neutralizing antibody, the enhanced IgM secretion by bupleuran 2IIc was reduced. When B cells were stimulated with bupleuran 2IIc, their IL-6 secretion and the transcription of IL-6 mRNA were enhanced. The enhanced IL-6 secretion by bupleuran 2IIc was also observed in B cell line, Y16 cell. When bupleuran 2IIc was digested with endo-alpha-(1-->4)-D-polygalacturonase, the resulting enzyme resistant carbohydrate portion, "ramified" region (PG-1) composed of rhamnogalacturonan core containing neutral sugar side chains showed potent IL-6 secretion-enhancing activity. These results suggest that the "ramified" region of bupleuran 2IIc stimulates the secretion of IL-6 as the active site, and the resulting IL-6 may partially contribute the enhancement of IgM secretion as an autocrine and/or paracrine mechanism.

Adjuvants, Immunologic↗
PubMed2001

Characteristics and significance of yeasts' involvement in cassava fermentation for 'fufu' production.

Six different strains of yeast, namely Candida krusei, C. tropicalis, Pichia saitoi, Saccharomyces cerevisiae, P. anomala and Zygosaccharomyces bailii were found present in cassava-fermenting water in the early part of the fermentation. The latter part of the fermentation was dominated in all cases by three strains of yeast namely C. krusei, C. tropicalis and Z. bailii. All the yeast strains exhibited amylolytic capabilities while none was able to produce cellulase. All the strains except Zygosaccharomyces spp. exhibited polygalacturonase activity, but only C. krusei was able to produce linamarase. In a study on the inter-relationships between C. krusei and Lactobacillus plantarum, the growth of the yeast strain was not enhanced in cassava by the presence of the lactic acid bacteria, but the growth of the L. plantarum strain was significantly enhanced when co-inoculated with C. krusei.

Candida↗
PubMed2003

The microbial ecology of cocoa bean fermentations in Indonesia.

Cocoa beans are the principal raw material of chocolate manufacture. The beans are subject to a microbial fermentation as the first stage in chocolate production. The microbial ecology of bean fermentation (Forastero and Trinitario cultivars) was investigated at three commercial fermentaries in East Java, Indonesia by determining the populations of individual species at 12-h intervals throughout the process. The first 2-3 days of fermentation were characterised by the successional growth of various species of filamentous fungi, yeasts, lactic acid bacteria and acetic acid bacteria. The principal species found were Penicillium citrinum, an unidentified basidiomycete, Kloeckera apis, Saccharomyces cerevisiae, Candida tropicalis, Lactobacillus cellobiosus, Lactobacillus plantarum and Acetobacter pasteurianus. The later stages of fermentation were dominated by the presence of Bacillus species, mostly, Bacillus pumilus and Bacillus licheniformis. Glucose, fructose, sucrose and citric acid of the bean pulp were utilised during fermentation, with the production of ethanol, acetic acid and lactic acid that diffused into the beans. The filamentous fungi were notable for their production of polygalacturonase activity and probably contributed to the degradation of bean pulp.

Bacillus↗